Soluble forms of low affinity fc gamma receptors, process for their identification and dosage, a corresponding dosage kit, and applications

ABSTRACT

Receptors, characterized by the fact that they consist of the product obtained by affinity chromatography on a column coupled with 3 G8 antibodies or lectins or polyclonal anti-receptor FcR antibodies of a biological fluid of human origin, then by gel permeation. The spectrum of said product, electrophoresis acrylamide gel in reducing condition, comprising a major band corresponding to a molecular mass of between 72000 and 76000 daltons, and a number of minor bands. According to its purified form, the receptor consists of a glycoprotein with a molecular mass of between 72000 and 76000 daltons, recognized by ELISA and Western Blotting by the monoclonal anti-Leu 11b antibody. Application of said receptors to diagnosis and to follow-up treatment of diseases involving Fc receptors (infectious diseases, diseases of the autoimmune system, rejection of transplants, cancer and myeloma and AIDS), as well as to the study of human polymorphisms.

The present invention relates to soluble forms of low affinity receptorsfor the Fc fragment of IgG molecules; the present invention also relatesto a method for the identification and assay of soluble forms of lowaffinity Fc gamma receptors, especially human soluble Fc receptors; italso relates to a kit of the reagents necessary for carrying out thismethod; it relates finally to the applications of this method for thediagnosis of pathological conditions in which the above-mentionedreceptors are involved, such as infectious diseases, autoimmunediseases, transplant rejections, human complex diseases, cancers,myelomas and acquired immune deficiency syndrome (AIDS), for thetherapeutic monitoring of the course of these conditions, as well as forthe study of human polymorphisms.

At the surface of many cells, in particular the cells of the immunesystem (macrophages, B lymphocytes, polymorphonuclear leukocytes, NKcells, and the like), there are receptors which permit a relationshipbetween antibodies or immunoglobulins and these cells are involved inimmunity. These receptors have an affinity for the Fc fragment ofantibodies (FcR). This Fc fragment corresponds to the portion ofimmunoglobulins not endowed with antibody activity. The importance ofthese receptors is considerable, since their mediation makes possiblethe recognition of foreign antigens (viruses, tumor cells and the like)and the initiation of a cytotoxic reaction, by T lymphocytes or humoral,with secretion of specific antibodies by B lymphocytes; phagocytosis,that is to say the extraction from tissues and the blood of particlesrecognized as foreign by the cells of the reticuloendothelial system(monocytes, macrophages); cell interactions between different cellsinvolved in immunity; the transplacental transfer of antibodies frommother to fetus; and the clearance of immune complexes whose pathogenicrole in many conditions is known.

The existence of a secretion of these receptors for the Fc fragment(FcR) by mouse T cell cultures activated by a specific activator hasbeen demonstrated. These Fc receptor molecules, secreted in vitro undervery special conditions of activation and culture (in particular, inmedium devoid of all serum, which has a deleterious effect on the cellsin culture), still possess the capacity to bind immunoglobulins throughtheir Fc fragment, and they have hence been christened "immunoglobulinbinding factor" (IBF). The existence of this type of IBF has beendemonstrated for three classes of immunoglobulins, IgG, IgA and IgE,namely the factors IgGBF, IgABF and IgEBF.

It has, moreover, been demonstrated that these IBF molecules arecapable, when they are in contact with B lymphocytes in culture, or evenmyeloma cells in vitro, of completely inhibiting the activation of thesecells and, as a result, the secretion of immunoglobulins by these B ormyeloma cells. The molecules in question are hence secreted by cellsbelonging to the immune system under artificial conditions of in vitroculture, but are nevertheless endowed with a functional capacity to bindantibodies and to bring about a substantial suppression of theactivation of the B lymphocyte system (lymphokine).

The existence of an Fc receptor for mouse IgG immunoglobulins (FcR) inmouse serum has been demonstrated. These molecules have been definedboth by their capacity to be recognized by a monoclonal antibodyspecific for mouse Fc gamma receptors (monoclonal antibody 2.4G2) andtheir functional capacity to be purified from immunoglobulin Fcfragments. This discovery is extremely important, since this lymphokineis secreted at a level which increases with age (level zero at birth,appearing at around the fifth day in newborn mice and becomingsystematically detectable at around the seventh day), that this levelincreases in proportion to the presence of infections or, moregenerally, that the immune system is stimulated (mice kept in amicrobe-free environment having strictly zero levels throughout theirlife), this level being, moreover, determined in a relative manner bygenetic factors within a population of homozygotic mice.

In addition, the existence has now been discovered, in particular inhuman serum, of a lymphokine having the capacity to bind to the Fcfragments of immunoglobulin, namely the circulating soluble serum formof low affinity Fc gamma receptors, also designated by the abbreviationFc-gamma RLo, or alternatively "CD16".

These receptors are present in biological fluids (in particular serum)in polymerized form, as could be observed by molecular weightmeasurements which, for example, yielded a value above approximately700,000 for the glycoprotein which has been shown to have a molecularweight in the monomer state of 72,000 to 76,000.

The subject of the present invention is a low affinity, soluble Fc γ Rtype III receptor (or CD16), consisting of a glycoprotein of molecularmass 72,000-76,000 daltons which is recognized in ELISA and WesternBlotting by the monoclonal antibody anti-Leu 11b.

The subject of the invention is also a low affinity, soluble Fc γ R typeIII receptor (or CD16), which consists of the product obtained byaffinity chromatography, on a column coupled to 3G8 antibodies or tolectins (for example Lens Culinaris Agglutinin (LCA), wheatgermagglutinin or concanavalin) or to anti-FcR receptor polyclonalantibodies, of a biological fluid of human origin, followed by gelfiltration, the spectrum of the said product, in acrylamide gelelectrophoresis under reducing conditions, containing a major bandcorresponding to a molecular mass of between 72,000 and 76,000 daltonsand a plurality of minor bands, of which the main ones correspond tomolecular masses, respectively, of:

between 64,000 and 68,000 daltons

between 51,000 and 55,000 daltons

between 42,000 and 46,000 daltons

between 33,000 and 37,000 daltons.

The invention also relates to an Fc γ R type III receptor essentiallycomprising the fraction of molecular mass 33,000-37,000 daltons, asappears in acrylamide gel electrophoresis in the presence of a reducingagent and a detergent agent such as sodium dodecyl sulfate (SDS).

The biological fluids to which the present invention relates are, interalia, serum and plasma fluids, cephalo-rachidian fluids, urines andascitic fluids.

A feature of all these receptors which may be mentioned is that, in theDot Blot technique, they recognize a rabbit anti-FcR receptor polyclonalantibody.

The invention finally relates to each of the fractions of the Fc γ Rtype III receptor, as are defined above, as well as to all possiblecombinations of these fractions.

The present invention also relates to a method for the identification,detection or assay of these lymphokines having the capacity to bind tothe Fc fragments of immunoglobulin, in particular the human serumsoluble Fc receptor.

The method according to the present invention for the identification orassay of soluble forms of the low affinity Fc γ R type III receptor (orCD16) consists in:

(a) binding, to a solid phase, a first antibody, which is a monoclonalor polyclonal antibody or alternatively a fraction of a monoclonal orpolyclonal antibody (for example a Fab fragment), directed against aconformational epitope of the Fc γ receptor to be identified or assayed;

(b) washing the said solid phase to remove the said first antibody whichis not coupled;

(c) incubating the sample containing the Fc γ receptor to be assayed inthe presence of the solid phase coated with the said first antibody;

(d) washing to remove the material not specifically bound to the saidfirst antibody;

(e) incubating, in the presence of the resulting solid phase, a secondantibody, which is a monoclonal or polyclonal antibody, or alternativelya fraction of a monoclonal or polyclonal antibody (for example a Fabfragment), and which is an anti-Fc receptor recognizing the samecategory of Fc receptors as the first antibody, but by a completelydifferent epitope;

(f) washing to remove the said second antibody not specifically bound;

(g) incubating, in the presence of the resulting solid phase, a thirdantibody, which is an antibody capable of specifically recognizing thesaid second antibody;

(h) washing to remove the third antibody not specifically bound; and

(i) assaying the third antibody bound, and deducing therefrom thequantity of Fc receptor initially present in the sample.

In the stage (a), the monoclonal antibody 3G8 is used, in particular, asthe first antibody. In the stage (e), a mouse IgM consisting of themonoclonal antibody anti-Leu 11b is used, in particular, as the secondantibody. In the stage (g), a polyclonal antibody, namely a goatanti-mouse IgM antibody, is used as the third antibody.

According to an especially preferred embodiment of the method accordingto the invention, in the stage (g), a third antibody labeled with anenzyme is employed and, in the stage (i), a colorimetric substrate forthe said enzyme is added and, after the colorimetric reaction has beenstopped, for example by adding aqueous sulfuric acid solution, thecolorimetric change is read, from which the quantity of Fc receptorsought is deduced. In effect, the colorimetric change is proportional tothe quantity of the third antibody and, as a result, proportional to thequantity of second antibody, and hence of Fc receptor initially presentin the sample.

Preferably, the enzyme is peroxidase, in particular horseradishperoxidase, and the colorimetric substrate for peroxidase isortho-phenylenediamine, in the presence of hydrogen peroxide, thecolorimetric reading being performed at 492 nm.

The incubation of the stage (a), for the binding of the first antibodyto the solid phase, is performed, for example, at a temperature of theorder of 4° C., for a period of time ranging from 8 to 12 hours.

As regards the incubations of the stages (c), (e) and (g), these areperformed, in particular, at room temperature, over a period of timeranging from 1 to 4 hours.

FIG. 7 of the attached drawing explains the assay according to theinvention. The lymphokine sought, which is assayed specifically by thismethod, contains a first site of capture by the first antibody (3G8),and a 10 second site of detection by the sequence αLeu 11b-goatanti-mouse IgM antibody and peroxidase.

The subject of the present invention is also the kit of the reagentsnecessary for carrying out this method, this kit comprising:

a solid support, in particular a microtitration plate, provided with afirst antibody which is a monoclonal or polyclonal antibody, oralternatively a fraction of a monoclonal or polyclonal antibody, andwhich is directed against a conformational epitope of the Fc γ receptorto be assayed;

a second antibody, which is a monoclonal or polyclonal antibody, oralternatively a fraction of a monoclonal or polyclonal antibody, andwhich is an anti-Fc receptor recognizing the same category of Fcreceptors as the first antibody, but by a completely different epitope;

a third antibody, which is an antibody capable of specificallyrecognizing the second antibody; and

a system for assay of said third antibody.

These three antibodies, as well as a system for assay of the thirdantibody, have been defined above.

As a detector of background, that is to say as a negative control,xenogenic sera (fetal calf, horse, goat) are used.

As a standard enabling provision to be made for a qualitative positivecontrol of the method, a positive known serum is used, and, in order tobe able to quantify the method, an extract of human polymorphonuclearleukocytes are used. These polymorphonuclear leukocytes are purifiedaccording to classical purification techniques, are counted and are thenlysed with a mild detergent which does not destroy the conformationalstructures of the polymorphonuclear cell receptors, which would nolonger be able to be recognized subsequently during the test with the3G8 antibody, and, knowing the number of Fc receptors perpolymorphonuclear cell, the number of polymorphonuclear cells at thestart and the different dilutions at which these polymorphonuclear cellsare tested, it is possible, by a simple relationship, to determine thecorrespondence between the soluble Fc receptors in the sera tested and atheoretical number of Fc receptors of polymorphonuclear cells detectedin the cell lysate. A lysate, purified according to classicaltechniques, of human polymorphonuclear cells, lysed exclusively with anaqueous solution of 30 mM octylthio beta-d-glucoside+5 mM DFP at 4° C.

The applications of this method are essentially diagnostic applicationsand applications for monitoring the therapeutic use of theselymphokines. The latter are, in effect, substances, the variations ofwhich are linked to the fine activation of the B lymphocyte systemand/or tho macrophage system and whose variation in level enables thestate of stimulation of this system to be determined. This is of majorimportance in infectious diseases, autoimmune diseases, transplantrejections, cancers and myelomas, and acquired immune deficiencysyndrome (AIDS). Moreover, after injection of the lymphokine (human Fcreceptor), it is possible, by means of this test, to assay thevariations in the serum levels and, as a result, to manage moreeffectively and with less toxicity the therapeutic capabilities of thislymphokine in human medicine. In effect, such a lymphokine is capable,like its in vitro homolog, of specifically inhibiting the secretion ofan immunoglobulin class and hence of halting an effector mechanism insome pathological conditions, such as the abovementioned diseases,without thereby, as a result of its specificity--in effect, a receptorfor IgG will bring about the inhibition of only IgG secretion--bringingabout an inhibition of the secretion of the other immunoglobulinclasses, hence enabling a resultant immunosuppression of the patient tobe avoided.

Likewise, by blocking the Fc fragments of immunoglobulins alreadyengaged in an adverse antigen-antibody reaction (as in the case ofantiplatelet autoantibodies in idiopathic thrombocytopenic purpura, forexample), this lymphokine may prevent the relationship between theseimmune complexes and the cells of the reticuloendothelial system whichwould normally have phagocytosed these antigens (platelets in the caseof idiopathic thrombocytopenic purpura), leading to the pathogeniceffect (thrombocytopenia in the chosen example).

The invention also relates to a medicinal product containing an Fc γ Rtype III receptor or at least one fraction which is a constituentthereof, as are defined above.

A particular embodiment of the assay method of the invention will bedescribed below.

(1) Preparation of the Microtitration Plates

The microtitration plates used are 96-well U-bottomed poly(vinyl)chloride plates (manufactured by Societe "Dynatech").

The monoclonal antibody 3G8 is introduced, on the basis of 3 μg perwell, in 100 μl of carbonate buffer at pH 9.6. The incubation lasts 8 to12 hours at a temperature of 4° C.

(2) Washing

The plates are washed 6 times with PBS (Phosphate Buffered Saline)containing 0.1% v/v of Tween 20. This washing solution, which will beused again in subsequent stages of this assay, will now be designatedmore simply by the term "PBS Tween".

(3) Deposition of the Sample to be Assayed

100 μl of the sample to be assayed, pure or diluted in PBS Tween whichhas been left for 4 hours at approximately 22° C. (room temperature),are deposited in each well.

(4) Washing

This washing is performed in the same manner as in the stage (2).

(5) Deposition of Anti-Leu-11b Antibody

80 ng of anti-Leu-11b antibody (source: Beckton-Dickinson), diluted in100 μl of PBS Tween, are deposited in each well. Incubation is performedfor 2 hours at approximately 22° C.

(6) Washing

This washing is performed in the same manner as in the stage (2).

(7) Deposition of Goat Anti-mouse IgM Antibody Conjugated to HorseradishPeroxidase

The abovementioned antibody (source: Jackson Immuno-ResearchLaboratories Inc.), diluted to 1/5,000 in PBS Tween producing in total100 μl per well, is introduced. Incubation is performed for 1 hour at22° C.

(8) Washing

This washing is performed in the same manner as in the stage (2).

(9) Deposition of the Colorimetric Substrate for Peroxidase

150 μl per well of citrate phosphate buffer containing 4 mg/ml ofortho-phenylenediamine and 0.8 μl/ml of hydrogen peroxide are deposited.

The colorimetric reaction is stopped by depositing 75 μl per well of 10%aqueous sulfuric acid solution, and the result is read at 492 nm in anELISA reading apparatus.

By means of the assay method according to the present invention, amolecule corresponding to a soluble serum form of the low affinityreceptor for the Fc fragment of IgG has been demonstrated in humanblood.

The characterization of the Fc γ R type III receptors of the inventionwill now be described.

A normal human serum (68 ml) is passed, in a first stage, through acolumn of Sepharose coupled to 3G8 monoclonal antibodies (2 mg of 3G8antibody per ml of Sepharose; total column volume: 1 ml), at roomtemperature. This column is then washed using 150 ml of a 0.1% strengthsolution of "Tween 20" in phosphate buffered saline solution, and thematerial adsorbed is then eluted using 1/2M acetic acid solution, thefragments having a volume of 0.5 ml. These fractions are immediatelyneutralized using 3M sodium bicarbonate solution. The serum, before andafter affinity chromatography, as well as the elution fractions, aretested for their content of soluble low affinity type III Fc receptor.The fractions which show immunological activity are then combined, and0.2 ml of this pool of fractions is subjected to gel filtration on acolumn of volume 25 ml of "Superose 6" (Pharmacia); this "Superose 6"column is equilibrated in 50 mM ammonium bicarbonate solution at pH 8.The fractions having a volume of 0.5 ml are collected and tested using adirect ELISA assay method. This ELISA method consists in testing theelution fractions without passing through the stage of capture using themonoclonal antibody 3G8. These fractions are affixed directly to a solidsupport of the 96-well PVC plate type, and are then, after washing,reacted with the monoclonal antibody anti-Leu 11b, followed, after 2hours, by a second anti-mouse IgM antibody labeled with peroxidase.

FIG. 1 of the attached drawing shows the results of this second stage ofthe purification, hence corresponding to the gel filtration. Two curvesare seen in this figure. The elution fraction numbers, as well as thetime elapsed (expressed in minutes) are plotted as abscissae, and theoptical density as seen on the reading of the wells in the direct ELISAmethod is plotted as ordinates. The curve in the form of a continuousline corresponds to the elution chromatogram of this gel filtration, andgives only the quantity of proteins without giving theircharacteristics. The second curve (∘--∘) represents the immunoreactivityof each of the elution fractions, in terms of purified soluble Fcreceptor. It is hence observed that the peak between the fractions 20and 25 bears virtually the whole of the purified soluble Fc type IIIreceptor type immunoreactivity.

The fractions 20 and 22, which are the richest in soluble Fc receptortype immunoreactivity since the optical density rises to around 2 units,were lyophilized, and they were subjected to acrylamide gelelectrophoresis in the presence of SDS and a reducing agent. Thesefractions are shown in FIG. 2, and show the existence of acharacteristic principal major band whose molecular weight is 72,000 to76,000 daltons, but also bands of somewhat minor importance at around66,000, 3,000, 43,000 and 35,000 daltons.

These bands are then transferred by horizontal flow onto anitrocellulose filter, according to the technique known as "WesternBlotting"; this cellulose nitrate paper is then reacted with themonoclonal antibody anti-Leu 11b and, after washing, this cellulosenitrate filter is reacted with anti-mouse IgM polyclonal antibodylabeled with alkaline phosphatase. This is shown in FIG. 3. It is foundthat only one of these bands actually reacts in the Western Blottingtechnique, and is hence still recognized by the anti-Leu 11b antibody;it is the major band at 72,000-76,000 daltons.

FIGS. 4 and 5 illustrate the results of adsorption experiments oncolumns of lectin. The experiment was as follows: 500 microliters ofnormal human serum were deposited twice in succession, at roomtemperature, in an equivalent volume of agarose coupled to differentlectins. In FIG. 4, the lectin in question is that known as LensCulinaris Agglutinin (or LCA). After the column eluant had beencollected, that is to say after everything not retained by the columnhad been collected, the Fc receptor reactivity was measured using theindirect ELISA technique as described above. The immunoreactivity of thesera before passage through these lectins (solid column) and of theeffluent, that is to say of the substances nor retained in the column,was tested, and an almost 90% adsorption of the material according tothe invention, representing the Fc receptor immunoreactivity, on thesecolumns is observed. This proves that this material is glycoprotein innature, and contains glucose and/or mannose residues.

The material adsorbed on this column was then eluted, that is to saydetached; this is shown in FIG. 5; this was carried out by applying onthe column a solution which enters into competition with the Fcreceptor, namely a solution which contains the sugar specific for thislectin. In this instance, the elution solution is a 0.5M α-methylmannoside solution. In this way, it was possible to elute the Fcreceptors from this lectin column; this is shown in FIG. 5. Theimmunoreactivity, as obtained using the classical sandwich ELISAtechnique, of the different elution fractions, which are numbered asabscissae, are shown in this figure, and the ordinates indicate thereactivity in ELISA in terms of optical density. It is seen that aclassical elution curve, with a peak around the second and thirdfraction, is indeed obtained. This confirms once more that the Fcreceptor according to the invention, as it circulates in the serum, isindeed a glycoprotein.

FIG. 6 illustrates the results of a so-called "Dot Blot" experiment. TheDot Blot technique is fully known and referred to in the literature.According to this technique, a cellulose nitrate filter is brought intocontact with a solution containing the antigens to be assayed. Theseantigens are bound to this cellulose nitrate paper by the application ofa vacuum on the other side of this porous filter. As a result of theaction of the vacuum, the antigen molecules become bound to thecellulose nitrate paper. This paper is then reacted with differentantibodies and enables colorimetric reactions to be observed by means ofthe use of enzyme-labeled antibodies.

In this case, there were three samples, which were tested in parallel:

on the bottom line, the sample is a cell extract, as also describedabove. This cell extract is tested at four dilutions, from the left tothe right of the figure, and shows a classical dilution curve with animmunoreactivity corresponding to the colorimetric reaction observed onthe nitrocellulose filter which decreases progressively in proportion tothe dilution.

On the middle line, in place of the cell extract, the sample is a serumconsidered to be positive from the results of the sandwich ELISAtechnique according to the invention. In this case also, it is foundthat the test shows a decrease in the optical density obtained,proportional to the dilution of serum which also goes from the left tothe right of the figure, hence showing that the reaction in question isequally as specific as that of the sandwich ELISA technique.

On the top line, a serum was tested which was considered to be negativefrom the results of the sandwich ELISA technique, that is to say, whenit is reacted in a first stage with capture by 3G8 on a PVC plate and inthe second instance with the system comprising anti-Leu 11b antibody andperoxidase-labeled anti-mouse IgM polyclonal antibody, which usuallygave a negative result, the presence was observed, in this negativeserum, of material antigenically recognized as an Fc receptor. The onlydifference here, compared with the sandwich ELISA technique, is, in thefirst place, that there is no capture stage, and hence no need for thisantigen to be recognized by the 3G8 monoclonal antibody, and in thesecond place, that the antibodies used for visualizing the Fc receptorare not the Leu 11b antibody and anti-mouse IgM antibody but, directly,a rabbit anti-Fc receptor polyclonal antibody, manufactured in thelaboratory by immunization of a rabbit with the purified Fc receptoraccording to the technique described in relation to FIG. 1, and aperoxidase-labeled rabbit anti-immunoglobulin antibody as secondantibody.

This shows that some sera considered to be negative by the use of the3G8/anti-Leu 11b system, are, however, soluble Fc type III receptorcarriers. This suggests that an antigen polymorphism exists, someindividuals having soluble plasma Fc receptors which are recognized by3G8/anti-Leu 11b, others having soluble serum Fc receptors which are notrecognized by the G8/anti-Leu 11b system.

By means of the assay and purification methods according to theinvention, a molecule is made available which can be used in man in thetreatment of different conditions:

1--Monoclonal secretions of immunoglobulins, such as multiple myeloma orKahler's disease.

2--Immune complex disease or diseases accompanied by the presence oftissue or serum immune complexes, such as lupus erythematosus and otherautoimmune diseases.

3--Lymphomas and leukemias, in particular Burkitt's lymphoma.

4--Bone marrow or organ (liver, kidney, heart, for example) transplantrejection, for curative or preventive purposes.

5--Acquired immune deficiencies and in particular during infection byHIV virus.

We claim:
 1. An isolated low affinity soluble Fc γ R type III receptor,which is a glycoprotein of molecular mass 72,000 to 76,000 daltons andwhich is immunologically recognized in ELISA and Western Blotting by themonoclonal antibody anti-Leu 11b.
 2. A purified low affinity soluble Fcγ R type III receptor which is at least substantially the same as theproduct obtained by subjecting a biological fluid of human origin toaffinity chromatography on a column coupled to a) 3G8 antibodies, b)lectins or c) anti-Fc R receptor polyclonal antibodies, eluting materialabsorbed on the column and gel filtering thus-obtained eluant, saidproduct having a spectrum, in acrylamide gel electrophoresis underreducing conditions, which has a major band corresponding to a molecularmass of between 72,000 and 76,000 daltons and a plurality of minorbands, the main ones corresponding to molecular masses, respectively,of:a) between 64,000 and 68,000 daltons, b) between 51,000 and 55,000daltons, c) between 42,000 and 46,000 daltons, and d) between 33,000 and37,000 daltons.
 3. A fraction of an Fc γ R type III receptor as claimedin claim 2 which has a molecular mass primarily between 33,000 and37,000 daltons, as appears in acrylamide gel electrophoresis in thepresence of a reducing agent and a detergent.
 4. A fraction of an Fc γ Rtype III receptor as claimed in claim 2 which has a molecular massprimarily between 64,000 and 68,000 daltons, as appears in acrylamidegel electrophoresis in the presence of a reducing agent and a detergent.5. A fraction of an Fc γ R type III receptor as claimed in claim 2 whichhas a molecular mass primarily between 51,000 and 55,000 daltons, asappears in acrylamide gel electrophoresis in the presence of a reducingagent and a detergent.
 6. A fraction of an Fc γ R type III receptor asclaimed in claim 2 which has a molecular mass primarily between 42,000and 46,000 daltons, as appears in a acrylamide gel electrophoresis inthe presence of a reducing agent and a detergent.
 7. An Fc γ R type IIIreceptor as claimed in claim 1 or claim 2 or a fraction of either whichcross-reacts with a rabbit anti-FcR receptor polyclonal antibody.
 8. Afraction of low affinity soluble Fc γ type III receptor, as claimed inclaim 1 or claim 2, in combination with one or more other fractions of alow affinity soluble Fc γ type III receptor or CD16, as claimed in claim1 or claim
 2. 9. A receptor as claimed in claim 1 or claim 2, and whichis in its native form.
 10. A receptor as claimed in claim 2, and whichis the same as the product obtained by the process set forth in claim 2.11. A kit of reagents, which comprises, in at least two separatecontainers:a) a solid support provided with a first antibody which isdirected against a conformational epitope of an Fc γ receptor, of claim1 or 2 to be assayed; the first antibody being a monoclonal antibody, apolyclonal antibody or a fraction of either; b) a second antibody whichis an anti-Fc receptor recognizing the same category of Fc receptors asthe first antibody, but by a completely different epitope; the secondantibody being a monoclonal antibody, a polyclonal antibody or afraction of either; c) a third antibody, which has a specific capabilityof recognizing the second antibody; and d) a system for assay of thethird antibody.
 12. A kit as claimed in claim 11, wherein the solidsupport is a microtitration plate.
 13. A kit as claimed in claim 12,wherein the first antibody is the monoclonal antibody 3G8 the secondantibody is a monoclonal antibody consisting of mouse IgM or anti-Leu11b antibody, and the third antibody is goat anti-mouse IgM polyclonalantibody.
 14. The kit as claimed in claim 11, wherein the system for theassay of the third antibody comprises an enzyme conjugated to the saidthird antibody, a colorimetric substrate associated with the said enzymeand a substance designed to stop the colorimetric reaction.
 15. A kit asclaimed in claim 14, wherein the enzyme is peroxidase, the substrateassociated with the said enzyme is ortho-phenylenediamine in thepresence of hydrogen peroxide, and the substance designed to stop thecolorimetric reaction is aqueous sulfuric acid solution.
 16. A kit asclaimed in claim 15, wherein the peroxidase in horseradish peroxidase.17. A method for identifying or for assay of a soluble form of lowaffinity Fc γ R type III receptor, which consists essentially of thefollowing steps:a) binding, to a solid phase, a first antibody, which isa monoclonal or polyclonal antibody or a fraction of a monoclonal orpolyclonal antibody, and which immunologically recognizes aconformational epitope of the Fc γ receptor to be identified or assayed;b) washing the solid phase to remove therefrom that portion of the firstantibody which is not bound thereto; c) incubating a sample, containingthe Fc γ receptor to be assayed, in the presence of said solid phasecoated with said first antibody; d) washing the resulting solid phase toremove therefrom material not specifically bound to the first antibody;e) incubating, in the presence of thus-washed solid phase, a secondantibody, which is a monoclonal or polyclonal antibody or a fraction ofa monoclonal or polyclonal antibody, and which is an anti-Fc receptorantibody which recognizes the same category of Fc receptors as the firstantibody, but by a completely different epitope; f) washingthus-obtained solid phase to remove therefrom that portion of the secondantibody which is not specifically bound thereto; g) incubating, in thepresence of thus-washed solid phase, a third antibody, which is capableof specifically recognizing said second antibody; h) washingthus-obtained solid phase to remove therefrom that portion of the thirdantibody which is not specifically bound thereto; i) assaying the thirdantibody bound to the thus-washed solid phase; and j) deducing therefromthe quantity of Fc receptor initially present in the sample.
 18. Amethod as claimed in claim 17, wherein the first antibody is monoclonalantibody 3G8.
 19. A method as claimed in claim 18, wherein the secondantibody is a mouse IgM consisting of antibody anti-Leu 11b.
 20. Amethod as claimed in claim 19, wherein the third antibody is a goatanti-mouse IgM polyclonal antibody.
 21. A method as claimed in one ofclaims 17 or 20 wherein a) the third antibody is labeled with an enzyme,b) a colorimetric substrate for the enzyme is reacted with the enzyme instep (i), c) after the colorimetric reaction has stopped, opticaldensity change is measured, and d) the quantity of Fc receptor sought isdeduced from the optical density change.
 22. A method as claimed inclaim 21, wherein the enzyme is horseradish peroxidase, the calorimetricsubstrate is orthophenylenediamine, in the presence of hydrogenperoxide, and the optical density is measured at 492 nm.
 23. A method asclaimed in one of claims 17 to 20 or in claim 22, wherein binding instep (a) is conducted at a temperature of about 4° C. for a period oftime ranging from 8 to 12 hours.
 24. A method as claimed in one of claim17 to 20 or in claim 22, wherein incubating in each of steps (c), (e)and (g) is effected at room temperature for a period of time rangingfrom 1 to 4 hours.